Project 3 Summary The goal of this program is to develop a vaccine for human immunodeficiency virus (HIV)-1 based on spread- deficient cytomegalovirus (CMV) vectors optimized for the induction of protective immune responses. Rhesus CMV (RhCMV)-vectored vaccines demonstrated unprecedented efficacy against highly virulent simian immunodeficiency virus (SIV), resulting in stringent control and clearance of SIV over time. Preliminary data suggest that this efficacy correlates with the induction of unconventional CD8+ T cells recognizing epitopes in the context of MHC-II and non-classical MHC-E molecules. In this project we will dissect the mechanisms by which strain 68-1 RhCMV achieves these unprecedented T cell responses to improve protection against SIV, and to design human CMV (HCMV)-based vectors that recapitulate the optimally protective immunological profile identified in monkeys. We will focus on understanding the function of RhCMV proteins and microRNAs (miRs) involved in MHC-E-restricted CD8+ T cell response priming, a likely candidate for mediating protection against SIV (see Project 1). Preliminary work has identified a number of RhCMV genes that, if deleted from strain 68-1 RhCMV vectors, abrogate MHC-E-restricted CD8+ T cell priming, resulting in vectors that exclusively elicit MHC-II-restricted T cells. As described in Project 1, these ?MHC-II-only? vectors will enable our determination of the contribution of MHC-II-, and indirectly, MHC-E-restricted responses to protection. Given that these genes are required for MHC-E-restricted epitope targeting, their characterization offers an opportunity to delineate the mechanisms responsible for priming this unusual type of CD8+ T cell response. A better understanding of the mechanisms by which RhCMV gene products control MHC-E-restricted CD8+ T cell priming will allow us to design HCMV vectors that elicit similar responses in humans. In Specific Aim 1, we will study the Rh67 gene, which encodes a protein containing the canonical, MHC-E-binding peptide VL9 required for intracellular transport of MHC-E in RhCMV-infected cells. We will determine whether the HCMV orthologue of Rh67, UL40, performs a similar function and we will mechanistically dissect the structural domains of Rh67 that are required for function, in particular the role of the embedded VL9 peptide, its position in the Rh67 protein, and non-VL9 protein sequence in priming MHC-E-restricted CD8+ T cells by both RhCMV and HCMV. In Specific Aim 2, we will test the hypothesis that viral miRs that alter vesicular trafficking in CMV- infected cells so as to create a viral assembly complex regulate peptide exchange of VL9-loaded MHC-E in this viral assembly compartment, thus promoting the induction of MHC-E-restricted CD8+ T cells. In Specific Aim 3, we will additionally study how G-protein coupled receptor (GPCR)-like proteins encoded in the Rh214-220 region promote MHC-E-restricted T cell responses and whether this function is conserved in the related HCMV proteins US28 and US27. We expect that this project will contribute to the generation of an HCMV/HIV vector that closely recapitulates the protective CD8+ T cell response profile elicited by optimized RhCMV/SIV vectors.